PROJECT SUMMARY The overall objectives of this NRSA individual fellowship are to identify and investigate new therapeutic targets for epithelial injury due to cigarette smoke in chronic obstructive lung disease (COPD), and to facilitate the development of essential skills that will allow the candidate to become an effective physician-scientist. The candidate and her mentors have constructed a rigorous training plan that will provide the foundation for a strong and successful academic career. The proposal addresses the fundamental biological processes of mitochondrial and metabolic regulation involved in cigarette smoking-related lung disease. Chronic obstructive pulmonary disease (COPD) is the 3rd leading cause of death in the US with cigarette smoke (CS) being the primary insult leading to disease. Despite ongoing mammalian research, no new biologic targets have been identified, and current therapies have limited effect on disease progression or mortality. We are leveraging the amoebozoan Dictyostelium discoideum to develop a comparative approach with human airway epithelial cells to identify new pathways and therapeutic strategies protective against CS. As an oxidative insult, CS results in morphologic and metabolic changes. We already found that CS leads to a relevant phenotype of growth suppression in Dictyostelium. Furthermore, using a cDNA library genetic selection from ~35,000 Dictyostelium cDNA transformants exposed to cigarette smoke extract (CSE), we identified adenine nucleotide translocase (ANT) as protective against CS-induced growth defects. ANT is a mitochondrial ADP/ATP transport protein that plays an active role in mitochondrial metabolism and energy homeostasis. Currently, the role of the ANT mitochondrial system in protecting the lung epithelium against cigarette-smoke induced injury remains unclear. The long-term goal of the proposal is to determine the mechanisms through which ANT is protective against CS injury and identify new potential therapeutic targets. In Aim 1, the candidate will decipher the effects of human ANT on cell viability and morphology in bronchial epithelial cells (HBEs) after cigarette smoke (CS). This will be accomplished by a combination of molecular expression tools, cell imaging, and genetic modification of these proteins to assess cell viability and structure. In Aim 2, the candidate will identify the metabolic and inflammatory mechanisms through which human ANT protects against CS injury in HBEs. This will be accomplished by evaluating real-time mitochondrial metabolism and cytokine changes due to ANT modulation in HBEs. The goal of these experiments is to leverage mammalian and Dictyostelium models to dissect the mitochondrial mechanisms leading to epithelial dysregulation in COPD. Completion of these aims will elucidate the role of the mitochondrial ANT in lung epithelium in response to CS, as a novel protective pathway in COPD. Furthermore, this work will lay groundwork for examination of new therapeutic strategies for COPD using translational in vivo models and an FDA-approved drug library.